COVID-19 mRNA vaccines induce robust levels of IgG but limited amounts of IgA within the oronasopharynx of young children.

BACKGROUND: Understanding antibody responses to SARS-CoV-2 vaccination is crucial for refining COVID-19 immunization strategies. Generation of mucosal immune responses, including mucosal IgA, could be of potential benefit to vaccine efficacy, yet limited evidence exists regarding the production of mucosal antibodies following the administration of current mRNA vaccines to young children. METHODS: We measured the levels of antibodies against SARS-CoV-2 from a cohort of children under 5 years of age (N=24) undergoing SARS-CoV-2 mRNA vaccination (serially collected, matched serum and saliva samples) or in a convenience sample of children under 5 years of age presenting to pediatric emergency department (nasal swabs, N=103). Further, we assessed salivary and nasal samples for the ability to induce SARS-CoV-2 spike-mediated neutrophil extracellular traps (NET) formation. RESULTS: Longitudinal analysis of post-vaccine responses in saliva revealed the induction of SARS-CoV-2 specific IgG but not IgA. Similarly, SARS-CoV-2 specific IgA was only observed in nasal samples obtained from previously infected children with or without vaccination, but not in vaccinated children without a history of infection. In addition, oronasopharyngeal samples obtained from children with prior infection were able to trigger enhanced spike-mediated NET formation, and IgA played a key role in driving this process. CONCLUSIONS: Despite the induction of specific IgG in the oronasal mucosa, current intramuscular vaccines have limited ability to generate mucosal IgA in young children. These results confirm the independence of mucosal IgA responses from systemic humoral responses following mRNA vaccination and suggest potential future vaccination strategies for enhancing mucosal protection in this young age group.

COVID-19 mRNA vaccines induce robust levels of IgG but limited amounts of IgA within the oronasopharynx of young children.

Background: Understanding antibody responses to SARS-CoV-2 vaccination is crucial for refining COVID-19 immunization strategies. Generation of mucosal immune responses, including mucosal IgA, could be of potential benefit to vaccine efficacy, yet limited evidence exists regarding the production of mucosal antibodies following the administration of current mRNA vaccines to young children. Methods: We measured the levels of antibodies against SARS-CoV-2 from a cohort of children under 5 years of age undergoing SARS-CoV-2 mRNA vaccination (serially collected, matched serum and saliva samples, N=116) or on convenience samples of children under 5 years of age presenting to a pediatric emergency department (nasal swabs, N=103). Further, we assessed salivary and nasal samples for the ability to induce SARS-CoV-2 spike-mediated neutrophil extracellular traps (NET) formation. Results: Longitudinal analysis of post-vaccine responses in saliva revealed the induction of SARS-CoV-2 specific IgG but not IgA. Similarly, SARS-CoV-2 specific IgA was only observed in nasal samples obtained from previously infected children with or without vaccination, but not in vaccinated children without a history of infection. In addition, oronasopharyngeal samples obtained from children with prior infection were able to trigger enhanced spike-mediated NET formation, and IgA played a key role in driving this process. Conclusions: Despite the induction of specific IgG in the oronasal mucosa, current intramuscular vaccines have limited ability to generate mucosal IgA in young children. These results confirm the independence of mucosal IgA responses from systemic humoral responses following mRNA vaccination and suggest potential future vaccination strategies for enhancing mucosal protection in this young age group.

An enhanced IL17 and muted type I interferon nasal epithelial cell state characterizes severe COVID-19 with fungal coinfection.

Recent case reports and epidemiological data suggest that fungal infections represent an underappreciated complication among people with severe COVID-19. However, the frequency of fungal colonization in patients with COVID-19 and associations with specific immune responses in the airways remain incompletely defined. We previously generated a single-cell RNA-sequencing data set characterizing the upper respiratory microenvironment during COVID-19 and mapped the relationship between disease severity and the local behavior of nasal epithelial cells and infiltrating immune cells. Our previous study, in agreement with findings from related human cohorts, demonstrated that a profound deficiency in host immunity, particularly in type I and type III interferon signaling in the upper respiratory tract, is associated with rapid progression to severe disease and worse clinical outcomes. We have now performed further analysis of this cohort and identified a subset of participants with severe COVID-19 and concurrent detection of Candida species-derived transcripts within samples collected from the nasopharynx and trachea. Here, we present the clinical characteristics of these individuals. Using matched single-cell transcriptomic profiles of these individuals' respiratory mucosa, we identify epithelial immune signatures suggestive of IL17 stimulation and anti-fungal immunity. Further, we observe a significant expression of anti-fungal inflammatory cascades in the nasal and tracheal epithelium of all participants who went on to develop severe COVID-19, even among participants without detectable genetic material from fungal pathogens. Together, our data suggest that IL17 stimulation-in part driven by Candida colonization-and blunted interferon signaling represent a common feature of severe COVID-19 infection. IMPORTANCE: In this paper, we present an analysis suggesting that symptomatic and asymptomatic fungal coinfections can impact patient disease progression during COVID-19 hospitalization. By looking into the presence of other pathogens and their effect on the host immune response during COVID-19 hospitalizations, we aim to offer insight into an underestimated scenario, furthering our current knowledge of determinants of severity that could be considered for future diagnostic and intervention strategies.

Humoral profiles of toddlers and young children following SARS-CoV-2 mRNA vaccination.

Although young children generally experience mild symptoms following infection with SARS-CoV-2, severe acute and long-term complications can occur. SARS-CoV-2 mRNA vaccines elicit robust immunoglobulin profiles in children ages 5 years and older, and in adults, corresponding with substantial protection against hospitalizations and severe disease. Whether similar immune responses and humoral protection can be observed in vaccinated infants and young children, who have a developing and vulnerable immune system, remains poorly understood. To study the impact of mRNA vaccination on the humoral immunity of infant, we use a system serology approach to comprehensively profile antibody responses in a cohort of children ages 6 months to 5 years who were vaccinated with the mRNA-1273 COVID-19 vaccine (25 µg). Responses are compared with vaccinated adults (100 µg), in addition to naturally infected toddlers and young children. Despite their lower vaccine dose, vaccinated toddlers elicit a functional antibody response as strong as adults, with higher antibody-dependent phagocytosis compared to adults, without report of side effects. Moreover, mRNA vaccination is associated with a higher IgG3-dependent humoral profile against SARS-CoV-2 compared to natural infection, supporting that mRNA vaccination is effective at eliciting a robust antibody response in toddlers and young children.

Enhanced production of eicosanoids in plasma and activation of DNA damage pathways in PBMCs are correlated with the severity of ancestral COVID-19 infection.

Background: Many questions remain unanswered regarding the implication of lipid metabolites in severe SARS-CoV-2 infections. By re-analyzed sequencing data from the nasopharynx of a previously published cohort, we found that alox genes, involved in eicosanoid synthesis, were up-regulated in high WHO score patients, especially in goblet cells. Herein, we aimed to further understand the roles played by eicosanoids during severe SARS-CoV-2 infection. Methods and findings: We performed a total fatty acid panel on plasma and bulk RNA-seq analysis on peripheral blood mononuclear cells (PBMCs) collected from 10 infected and 10 uninfected patients. Univariate comparison of lipid metabolites revealed that lipid metabolites were increased in SARS-CoV-2 patients including the lipid mediators Arachidonic Acid (AA) and Eicosapentaenoic Acid (EPA). AA, EPA and the fatty acids Docosahexaenoic acid (DHA) and Docosapentaenoic acid (DPA), were positively correlated to WHO disease severity score. Transcriptomic analysis demonstrated that COVID-19 patients can be segregated based on WHO scores. Ontology, KEGG and Reactome analysis identified pathways enriched for genes related to innate immunity, interactions between lymphoid and nonlymphoid cells, interleukin signaling and, cell cycling pathways. Conclusions: Our study offers an association between nasopharynx mucosa eicosanoid genes expression, specific serum inflammatory lipids and, subsequent DNA damage pathways activation in PBMCs to severity of COVID-19 infection.

The IL-10 receptor inhibits cell extrinsic signals necessary for STAT1-dependent macrophage accumulation during colitis.

The loss of IL-10R function leads to severe early onset colitis and, in murine models, is associated with the accumulation of immature inflammatory colonic macrophages. We have shown that IL-10R-deficient colonic macrophages exhibit increased STAT1-dependent gene expression, suggesting that IL-10R-mediated inhibition of STAT1 signaling in newly recruited colonic macrophages might interfere with the development of an inflammatory phenotype. Indeed, STAT1-/- mice exhibit defects in colonic macrophage accumulation after Helicobacter hepaticus infection and IL-10R blockade, and this was phenocopied in mice lacking IFNγR, an inducer of STAT1 activation. Radiation chimeras demonstrated that reduced accumulation of STAT1-deficient macrophages was based on a cell-intrinsic defect. Unexpectedly, mixed radiation chimeras generated with both wild-type and IL-10R-deficient bone marrow indicated that rather than directly interfering with STAT1 function, IL-10R inhibits the generation of cell extrinsic signals that promote the accumulation of immature macrophages. These results define the essential mechanisms controlling the inflammatory macrophage accumulation in inflammatory bowel diseases.

Severe COVID-19 is associated with fungal colonization of the nasopharynx and potent induction of IL-17 responses in the nasal epithelium.

Recent case reports and epidemiological data suggest fungal infections represent an under-appreciated complication among people with severe COVID-19. However, the frequency of fungal colonization in patients with COVID-19 and associations with specific immune responses in the airways remain incompletely defined. We previously generated a single-cell RNA-sequencing (scRNA-seq) dataset characterizing the upper respiratory microenvironment during COVID-19, and mapped the relationship between disease severity and the local behavior of nasal epithelial cells and infiltrating immune cells. Our study, in agreement with findings from related human cohorts, demonstrated that a profound deficiency in host immunity, particularly in type I and type III interferon signaling in the upper respiratory tract, is associated with rapid progression to severe disease and worse clinical outcomes. We have now performed further analysis of this cohort and identified a subset of participants with severe COVID-19 and concurrent detection of Candida species-derived transcripts within samples collected from the nasopharynx and trachea. Here, we present the clinical characteristics of these individuals, including confirmatory diagnostic testing demonstrating elevated serum (1, 3)-ß-D-glucan and/or confirmed fungal culture of the predicted pathogen. Using matched single-cell transcriptomic profiles of these individuals' respiratory mucosa, we identify epithelial immune signatures suggestive of IL-17 stimulation and anti-fungal immunity. Further, we observe significant expression of anti-fungal inflammatory cascades in the nasal and tracheal epithelium of all participants who went on to develop severe COVID-19, even among participants without detectable genetic material from fungal pathogens. Together, our data suggests that IL-17 stimulation - in part driven by Candida colonization - and blunted type I/III interferon signaling represents a common feature of severe COVID-19 infection.

Monocytes transition to macrophages within the inflamed vasculature via monocyte CCR2 and endothelial TNFR2.

Monocytes undergo phenotypic and functional changes in response to inflammatory cues, but the molecular signals that drive different monocyte states remain largely undefined. We show that monocytes acquire macrophage markers upon glomerulonephritis and may be derived from CCR2+CX3CR1+ double-positive monocytes, which are preferentially recruited, dwell within glomerular capillaries, and acquire proinflammatory characteristics in the nephritic kidney. Mechanistically, the transition to immature macrophages begins within the vasculature and relies on CCR2 in circulating cells and TNFR2 in parenchymal cells, findings that are recapitulated in vitro with monocytes cocultured with TNF-TNFR2-activated endothelial cells generating CCR2 ligands. Single-cell RNA sequencing of cocultures defines a CCR2-dependent monocyte differentiation path associated with the acquisition of immune effector functions and generation of CCR2 ligands. Immature macrophages are detected in the urine of lupus nephritis patients, and their frequency correlates with clinical disease. In conclusion, CCR2-dependent functional specialization of monocytes into macrophages begins within the TNF-TNFR2-activated vasculature and may establish a CCR2-based autocrine, feed-forward loop that amplifies renal inflammation.

CCR2 promotes monocyte recruitment and intestinal inflammation in mice lacking the interleukin-10 receptor.

Macrophages are a heterogeneous population of mononuclear phagocytes abundantly distributed throughout the intestinal compartments that adapt to microenvironmental specific cues. In adult mice, the majority of intestinal macrophages exhibit a mature phenotype and are derived from blood monocytes. In the steady-state, replenishment of these cells is reduced in the absence of the chemokine receptor CCR2. Within the intestine of mice with colitis, there is a marked increase in the accumulation of immature macrophages that demonstrate an inflammatory phenotype. Here, we asked whether CCR2 is necessary for the development of colitis in mice lacking the receptor for IL10. We compared the development of intestinal inflammation in mice lacking IL10RA or both IL10RA and CCR2. The absence of CCR2 interfered with the accumulation of immature macrophages in IL10R-deficient mice, including a novel population of rounded submucosal Iba1+ cells, and reduced the severity of colitis in these mice. In contrast, the absence of CCR2 did not reduce the augmented inflammatory gene expression observed in mature intestinal macrophages isolated from mice lacking IL10RA. These data suggest that both newly recruited CCR2-dependent immature macrophages and CCR2-independent residual mature macrophages contribute to the development of intestinal inflammation observed in IL10R-deficient mice.

Utilizing a reductionist model to study host-microbe interactions in intestinal inflammation.

BACKGROUND: The gut microbiome is altered in patients with inflammatory bowel disease, yet how these alterations contribute to intestinal inflammation is poorly understood. Murine models have demonstrated the importance of the microbiome in colitis since colitis fails to develop in many genetically susceptible animal models when re-derived into germ-free environments. We have previously shown that Wiskott-Aldrich syndrome protein (WASP)-deficient mice (Was-/-) develop spontaneous colitis, similar to human patients with loss-of-function mutations in WAS. Furthermore, we showed that the development of colitis in Was-/- mice is Helicobacter dependent. Here, we utilized a reductionist model coupled with multi-omics approaches to study the role of host-microbe interactions in intestinal inflammation. RESULTS: Was-/- mice colonized with both altered Schaedler flora (ASF) and Helicobacter developed colitis, while those colonized with either ASF or Helicobacter alone did not. In Was-/- mice, Helicobacter relative abundance was positively correlated with fecal lipocalin-2 (LCN2), a marker of intestinal inflammation. In contrast, WT mice colonized with ASF and Helicobacter were free of inflammation and strikingly, Helicobacter relative abundance was negatively correlated with LCN2. In Was-/- colons, bacteria breach the mucus layer, and the mucosal relative abundance of ASF457 Mucispirillum schaedleri was positively correlated with fecal LCN2. Meta-transcriptomic analyses revealed that ASF457 had higher expression of genes predicted to enhance fitness and immunogenicity in Was-/- compared to WT mice. In contrast, ASF519 Parabacteroides goldsteinii's relative abundance was negatively correlated with LCN2 in Was-/- mice, and transcriptional analyses showed lower expression of genes predicted to facilitate stress adaptation by ASF519 in Was-/-compared to WT mice. CONCLUSIONS: These studies indicate that the effect of a microbe on the immune system can be context dependent, with the same bacteria eliciting a tolerogenic response under homeostatic conditions but promoting inflammation in immune-dysregulated hosts. Furthermore, in inflamed environments, some bacteria up-regulate genes that enhance their fitness and immunogenicity, while other bacteria are less able to adapt and decrease in abundance. These findings highlight the importance of studying host-microbe interactions in different contexts and considering how the transcriptional profile and fitness of bacteria may change in different hosts when developing microbiota-based therapeutics. Video abstract.

Impaired local intrinsic immunity to SARS-CoV-2 infection in severe COVID-19.

SARS-CoV-2 infection can cause severe respiratory COVID-19. However, many individuals present with isolated upper respiratory symptoms, suggesting potential to constrain viral pathology to the nasopharynx. Which cells SARS-CoV-2 primarily targets and how infection influences the respiratory epithelium remains incompletely understood. We performed scRNA-seq on nasopharyngeal swabs from 58 healthy and COVID-19 participants. During COVID-19, we observe expansion of secretory, loss of ciliated, and epithelial cell repopulation via deuterosomal cell expansion. In mild and moderate COVID-19, epithelial cells express anti-viral/interferon-responsive genes, while cells in severe COVID-19 have muted anti-viral responses despite equivalent viral loads. SARS-CoV-2 RNA+ host-target cells are highly heterogenous, including developing ciliated, interferon-responsive ciliated, AZGP1high goblet, and KRT13+ "hillock"-like cells, and we identify genes associated with susceptibility, resistance, or infection response. Our study defines protective and detrimental responses to SARS-CoV-2, the direct viral targets of infection, and suggests that failed nasal epithelial anti-viral immunity may underlie and precede severe COVID-19.

Increased ACE2 Levels and Mortality Risk of Patients With COVID-19 on Proton Pump Inhibitor Therapy.

INTRODUCTION: Proton pump inhibitor (PPI) use was recently reported to be associated with increased severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and worse clinical outcomes. The underlying mechanism(s) for this association are unclear. METHODS: We performed a prospective study of hospitalized coronavirus disease 2019 (COVID-19) patients and COVID-negative controls to understand how PPI use may affect angiotensin-converting enzyme 2 (ACE2) expression and stool SARS-CoV-2 RNA. Analysis of a retrospective cohort of hospitalized patients with COVID-19 from March 15, 2020 to August 15, 2020 in 6 hospitals was performed to evaluate the association of PPI use and mortality. Covariates with clinical relevance to COVID-19 outcomes were included to determine predictors of in-hospital mortality. RESULTS: Control PPI users had higher salivary ACE2 mRNA levels than nonusers, 2.39 ± 1.15 vs 1.22 ± 0.92 (P = 0.02), respectively. Salivary ACE2 levels and stool SARS-CoV-2 RNA detection rates were comparable between users and nonusers of PPI. In 694 hospitalized patients with COVID-19 (age = 58 years, 46% men, and 65% black), mortality rate in PPI users and nonusers was 30% (68/227) vs 12.1% (53/439), respectively. Predictors of mortality by logistic regression were PPI use (adjusted odds ratio [aOR] = 2.72, P < 0.001), age (aOR = 1.66 per decade, P < 0.001), race (aOR = 3.03, P = 0.002), cancer (aOR = 2.22, P = 0.008), and diabetes (aOR = 1.95, P = 0.003). The PPI-associated mortality risk was higher in black patients (aOR = 4.16, 95% confidence interval: 2.28-7.59) than others (aOR = 1.62, 95% confidence interval: 0.82-3.19, P = 0.04 for interaction). DISCUSSION: COVID-negative PPI users had higher salivary ACE2 expression. PPI use was associated with increased mortality risk in patients with COVID-19, particularly African Americans.

Variants in STXBP3 are Associated with Very Early Onset Inflammatory Bowel Disease, Bilateral Sensorineural Hearing Loss and Immune Dysregulation.

BACKGROUND AND AIMS: Very early onset inflammatory bowel disease [VEOIBD] is characterized by intestinal inflammation affecting infants and children less than 6 years of age. To date, over 60 monogenic aetiologies of VEOIBD have been identified, many characterized by highly penetrant recessive or dominant variants in underlying immune and/or epithelial pathways. We sought to identify the genetic cause of VEOIBD in a subset of patients with a unique clinical presentation. METHODS: Whole exome sequencing was performed on five families with ten patients who presented with a similar constellation of symptoms including medically refractory infantile-onset IBD, bilateral sensorineural hearing loss and, in the majority, recurrent infections. Genetic aetiologies of VEOIBD were assessed and Sanger sequencing was performed to confirm novel genetic findings. Western analysis on peripheral blood mononuclear cells and functional studies with epithelial cell lines were employed. RESULTS: In each of the ten patients, we identified damaging heterozygous or biallelic variants in the Syntaxin-Binding Protein 3 gene [STXBP3], a protein known to regulate intracellular vesicular trafficking in the syntaxin-binding protein family of molecules, but not associated to date with either VEOIBD or sensorineural hearing loss. These mutations interfere with either intron splicing or protein stability and lead to reduced STXBP3 protein expression. Knock-down of STXBP3 in CaCo2 cells resulted in defects in cell polarity. CONCLUSION: Overall, we describe a novel genetic syndrome and identify a critical role for STXBP3 in VEOIBD, sensorineural hearing loss and immune dysregulation.

Impaired local intrinsic immunity to SARS-CoV-2 infection in severe COVID-19.

Infection with SARS-CoV-2, the virus that causes COVID-19, can lead to severe lower respiratory illness including pneumonia and acute respiratory distress syndrome, which can result in profound morbidity and mortality. However, many infected individuals are either asymptomatic or have isolated upper respiratory symptoms, which suggests that the upper airways represent the initial site of viral infection, and that some individuals are able to largely constrain viral pathology to the nasal and oropharyngeal tissues. Which cell types in the human nasopharynx are the primary targets of SARS-CoV-2 infection, and how infection influences the cellular organization of the respiratory epithelium remains incompletely understood. Here, we present nasopharyngeal samples from a cohort of 35 individuals with COVID-19, representing a wide spectrum of disease states from ambulatory to critically ill, as well as 23 healthy and intubated patients without COVID-19. Using standard nasopharyngeal swabs, we collected viable cells and performed single-cell RNA-sequencing (scRNA-seq), simultaneously profiling both host and viral RNA. We find that following infection with SARS-CoV-2, the upper respiratory epithelium undergoes massive reorganization: secretory cells diversify and expand, and mature epithelial cells are preferentially lost. Further, we observe evidence for deuterosomal cell and immature ciliated cell expansion, potentially representing active repopulation of lost ciliated cells through coupled secretory cell differentiation. Epithelial cells from participants with mild/moderate COVID-19 show extensive induction of genes associated with anti-viral and type I interferon responses. In contrast, cells from participants with severe lower respiratory symptoms appear globally muted in their anti-viral capacity, despite substantially higher local inflammatory myeloid populations and equivalent nasal viral loads. This suggests an essential role for intrinsic, local epithelial immunity in curbing and constraining viral-induced pathology. Using a custom computational pipeline, we characterized cell-associated SARS-CoV-2 RNA and identified rare cells with RNA intermediates strongly suggestive of active replication. Both within and across individuals, we find remarkable diversity and heterogeneity among SARS-CoV-2 RNA+ host cells, including developing/immature and interferon-responsive ciliated cells, KRT13+ "hillock"-like cells, and unique subsets of secretory, goblet, and squamous cells. Finally, SARS-CoV-2 RNA+ cells, as compared to uninfected bystanders, are enriched for genes involved in susceptibility (e.g., CTSL, TMPRSS2) or response (e.g., MX1, IFITM3, EIF2AK2) to infection. Together, this work defines both protective and detrimental host responses to SARS-CoV-2, determines the direct viral targets of infection, and suggests that failed anti-viral epithelial immunity in the nasal mucosa may underlie the progression to severe COVID-19.

Characterizing T cell subsets in the nasal mucosa of children with acute respiratory symptoms.

BACKGROUND: In infants admitted to an ICU with respiratory failure, there is an association between the ratio of CD8+ to CD4+ T cells within the upper respiratory tract and disease severity. Whether this ratio is associated with respiratory disease severity within children presenting to a pediatric emergency department is not known. METHODS: We studied a convenience sample of 63 children presenting to a pediatric emergency department with respiratory symptoms. T cell subsets in the nasal mucosa were analyzed by flow cytometry. We compared CD4+ and CD8+ T cells subsets in these samples and analyzed the proportion of these subsets that expressed markers associated with tissue residency. RESULTS: We were able to identify major subsets of CD8 and CD4 T cells within the nasal mucosa using flocked swabs. We found no difference in the ratio CD8+ to CD4+ T cells in children with upper or lower respiratory illness. A positive association between tissue-resident memory T cell frequency and patient age was identified. CONCLUSIONS: In our patient populations, the CD8+:CD4+ ratio was not associated with disease severity. The majority of T cells collected on nasal swabs are antigen experienced, and there is an association between the frequency of tissue-resident T cells and age. IMPACT: Immune cell populations from the nasal mucosa can be captured using flocked nasal swabs and analyzed by flow cytometry. Nasal CD8+:CD4+ ratio does not predict respiratory illness severity in children presenting to the emergency department. The frequency of CD8+ and CD4+ resident memory T cells within the nasal mucosa increases with age.

Effects of Colonization of Gnotobiotic Swiss Webster Mice with Helicobacter bilis.

Helicobacter bilis (Hb) causes hepatitis in some strains of inbred mice. The current study confirmed that Hb directly causes portal hepatitis in outbred gnotobiotic Swiss Webster (SW) mice, as we previously reported for conventional SW mice. Hbmonoassociated SW mice also developed mild enterocolitis, expanded gut-associated lymphoid tissue (GALT), and tertiary lymphoid tissue in the lower bowel. At 1 and 10 mo after infection, Hb-induced GALT hyperplasia exhibited well-organized, ectopic germinal centers with increased mononuclear cell apoptosis, MHC class II antigen presentation, and pronounced endothelial venule formation, consistent with features of tertiary lymphoid tissue. In the lower bowel, Hb induced mainly B220+ cells as well as CD4+ IL17+, CD4+ IFNγ+, and CD4+ FoxP3+ regulatory T cells and significantly increased IL10 mRNA expression. This gnotobiotic model confirmed that Hb causes portal hepatitis in outbred SW mice but stimulated GALT with an antiinflammatory bias. Because Hb had both anti- and proinflammatory effects on GALT, it should be considered a 'pathosymbiont provocateur' and merits further evaluation in mouse models of human disease.

Generation of protective pneumococcal-specific nasal resident memory CD4+ T cells via parenteral immunization.

The generation of tissue-resident memory T cells (TRM) is an essential aspect of immunity at mucosal surfaces, and it has been suggested that preferential generation of TRM is one of the principal advantages of mucosally administered vaccines. We have previously shown that antigen-specific, IL-17-producing CD4+ T cells can provide capsular antibody-independent protection against nasal carriage of Streptococcus pneumoniae; but whether pneumococcus-responsive TRM are localized within the nasal mucosa and are sufficient for protection from carriage has not been determined. Here, we show that intranasal administration of live or killed pneumococci to mice generates pneumococcus-responsive IL-17A-producing CD4+ mucosal TRM. Furthermore, we show that these cells are sufficient to mediate long-lived, neutrophil-dependent protection against subsequent pneumococcal nasal challenge. Unexpectedly, and in contrast with the prevailing paradigm, we found that parenteral administration of killed pneumococci also generates protective IL-17A+CD4+ TRM in the nasal mucosa. These results demonstrate a critical and sufficient role of TRM in prevention of pneumococcal colonization, and further that these cells can be generated by parenteral immunization. Our findings therefore have important implications regarding the generation of immune protection at mucosal surfaces by vaccination.

Alpha kinase 1 controls intestinal inflammation by suppressing the IL-12/Th1 axis.

Inflammatory bowel disease (IBD) are heterogenous disorders of the gastrointestinal tract caused by a spectrum of genetic and environmental factors. In mice, overlapping regions of chromosome 3 have been associated with susceptibility to IBD-like pathology, including a locus called Hiccs. However, the specific gene that controls disease susceptibility remains unknown. Here we identify a Hiccs locus gene, Alpk1 (encoding alpha kinase 1), as a potent regulator of intestinal inflammation. In response to infection with the commensal pathobiont Helicobacter hepaticus (Hh), Alpk1-deficient mice display exacerbated interleukin (IL)-12/IL-23 dependent colitis characterized by an enhanced Th1/interferon(IFN)-γ response. Alpk1 controls intestinal immunity via the hematopoietic system and is highly expressed by mononuclear phagocytes. In response to Hh, Alpk1-/- macrophages produce abnormally high amounts of IL-12, but not IL-23. This study demonstrates that Alpk1 promotes intestinal homoeostasis by regulating the balance of type 1/type 17 immunity following microbial challenge.

WASP-mediated regulation of anti-inflammatory macrophages is IL-10 dependent and is critical for intestinal homeostasis.

Mutations in Wiskott-Aldrich syndrome protein (WASP) cause autoimmune sequelae including colitis. Yet, how WASP mediates mucosal homeostasis is not fully understood. Here we show that WASP-mediated regulation of anti-inflammatory macrophages is critical for mucosal homeostasis and immune tolerance. The generation and function of anti-inflammatory macrophages are defective in both human and mice in the absence of WASP. Expression of WASP specifically in macrophages, but not in dendritic cells, is critical for regulation of colitis development. Importantly, transfer of WT anti-inflammatory macrophages prevents the development of colitis. DOCK8-deficient macrophages phenocopy the altered macrophage properties associated with WASP deficiency. Mechanistically, we show that both WASP and DOCK8 regulates macrophage function by modulating IL-10-dependent STAT3 phosphorylation. Overall, our study indicates that anti-inflammatory macrophage function and mucosal immune tolerance require both WASP and DOCK8, and that IL-10 signalling modulates a WASP-DOCK8 complex.

Macrophage dysfunction initiates colitis during weaning of infant mice lacking the interleukin-10 receptor.

Infants with defects in the interleukin 10 receptor (IL10R) develop very early onset inflammatory bowel disease. Whether IL10R regulates lamina propria macrophage function during infant development in mice and whether macrophage-intrinsic IL10R signaling is required to prevent colitis in infancy is unknown. Here we show that although signs of colitis are absent in IL10R-deficient mice during the first two weeks of life, intestinal inflammation and macrophage dysfunction begin during the third week of life, concomitant with weaning and accompanying diversification of the intestinal microbiota. However, IL10R did not directly regulate the microbial ecology during infant development. Interestingly, macrophage depletion with clodronate inhibited the development of colitis, while the absence of IL10R specifically on macrophages sensitized infant mice to the development of colitis. These results indicate that IL10R-mediated regulation of macrophage function during the early postnatal period is indispensable for preventing the development of murine colitis.